lecture 18

Question 1

What is transgenesis?

Answer 1

the process of transferring genes to animals

Question 2

What is a transgenic animal?

Answer 2

an animal that carries a transgene

Question 3

What is a transgene?

Answer 3

a foreign gene

Question 4

What is a chimera?

Answer 4

an organism carrying cell populations from two or more different embryos of the same or different strains

• often use coat colour to see whether you have made your chimera

Question 5

What does early mammalian development involve?

Answer 5

• fertilised egg
• division of blastomeres
• start to pack together tightly –> formation of morula - it pushes cells inside and it pushes cells on the outside, so you’re left with some cells inside, starts to form these cells in the middle cells on the outside –> development of the blastocyst
• takes about 3/4 days in the mouse, 7 days in humans
• outside cells of the blastocyst are the trophoblast –> placenta
• inner cell mass = cells that form the embryo/human
• about 16 cells at the early stage
• before the blastocyst has implanted

Question 6

How do we generate chimeras?

Answer 6

• you fertilise an egg in the oviduct and it travels down the oviduct while it’s dividing
• once it gets to the uterus it is a blastocyst and it’s waiting to implant into the uterine wall
• you can flush the oviducts before they’ve implanted and you can collect these blastocysts
• culture them and take the ICM cells and they expand in culture –> can go for 30 odd years
• it is when cells are in culture that they can be genetically modified, make alterations of our choice
• inject selected ES cells from black mouse into blastocyst from white mouse (into the blastoceal cavity)
• only have to add between 10 and 12 cells into that cavity in order to generate a chimera
• these cells will now contribute to the organism –> divide etc
• when injected into donor blastocyst, ES cells contribute to all tissues of the resulting offspring – models of human disease

Question 7

What are the important steps in the gene-targeting techique?

Answer 7

a. generate the gene-targeting construct
- a piece of genome that you’ve extracted from the mouse, 2-3kb on either side of the region that you want to play with
- these side sequences are very important for driving homologous recombination
b. transfect construct into ES cells
- liposomes, electrophoration (most common, temporary holes in the membrane)
- can recombine and displace the region that is already there
c. select positively gene-targeted ES cells
- need to only take the cells that have taken up the construct
- doesn’t work the same way as with mammal ES cells
- use other selectable markers, antibiotics in particular that have been generated for use in ES cells
- after a period of time only the colonies that have taken up the construct will grow
d. inject gene-targeted cells into blastocysts
e. transfer blastocysts to uterus of foster mothers
f. genotype offspring
g. cross heterozygotes to breed to homozygosity
- necessary to create a null mutation

Question 8

What is homologous recombination?

Answer 8

• an exchange between similar genetic material

Question 9

What is heterologous recombination?

Answer 9

• an exchange between different genetic material

- random integration

Question 10

What are selectable markers for positive and negative selection?

Answer 10

• neomycin gene (NEO): G418 inhibits protein synthesis, Neo inactivates G418 (+)
• thymidine kinase gene (TK): ganciclovir nucleoside analog, TK converts ganciclovir into toxic product (-)
• flanking homologous sequences on either side, exon 1, neo, exon 1, TK downstream of flanking homologous sequence
• if you just put the neo cassette in you can’t tell if it is homologous or non-homologous
• if you add TK and it’s still present –> it will kill the cell that has taken it up
• only taken up in non-homologous recombination (not correct in all cases but a nifty trick)
• random integration - whole thing
• assumption is that in homologous recombination the whole construct does not go in –> the TK falls off
• non homologous will die in ganciclovir (even though resistant to G418)
• homologous –> Ganciclovir resistant –> cells live

Question 11

What do you see in an analysis of DNA extracted from offspring following heterozygote crossing?

Answer 11

• gene locus containing gene mutation by insertion of neomycin gene is longer than for wildtype locus
• using PCR primers that bind to both ends of gene locus can amplify DNA
• primers are the same for mutated and Wildtype locus
• wt/wt would have one band, shorter
• wt/m two bands
• m/m one band (size of mutated locus)

Question 12

What are knockouts?

Answer 12

• gene-targeting (precise location)
• loss of function
• null mutant
• no functional protein

Question 13

What are knockins?

Answer 13

• gene-targeting
• modification
• may be functional protein
• may be other species gene (protein)

Question 14

What are the applications of transgenics?

Answer 14

a. to study the function of a gene - create KO
b. generate models of human disease

does p53 have a role in cancer?
normal
- p53 levels very low
- prevents S-phase of cell cycle if DNA damaged

knockout p53 -/- mice

• mice normal, develop cancer by 3 months
• p53 mutations in human cancers
• model of human disease
• test new: drugs, vaccines, gene-therapy, etc

Question 15

What if you create a KO and discover it has functions in many organs? How can you study the effect of the KO in one organ only?

Answer 15

tissue-specific gene-targeting

cre-lox system: tissue-specific gene-targeting
- removes DNA from between two specific sequences

Animal one: transgenic expresses Cre recombinase only in lung

• lung specific promoter/Cre recombinase gene
• therefore this enzyme is only expressed in the lung

Animal two: floxed gene created by gene targeting (flanked by LoxP site)

first two animals are totally normal

• cross the animals
• floxed gene meets Cre recombinase only in lung cells

Question 16

What are applications of the cre-lox system?

Answer 16

normal:
Tcf 21 (Pod1, capsulin) - what is role?

KO - animals die within 5 mins of birth due to defects in lungs and other organs

• suggest Tcf21 has an important role in organ development
• can study the tissues to examine defects at a cellular level
• can study target genes by microarray or transcriptome analysis – this will allow you to understand what genes are turned on or off by Tcf21

since Tcf 21 -/- animals die within 5 mins, difficult to study specific role of Tcf21 in kidney development

generate two animals:

1. kidney specific promoter - Cre recombinase. promoter activare during late embryonic development (but could theoretically test at any stage of development with unique promoters)
2. floxed Tcf21

cross animals

• Tcf21 only KO in kidney
• can now study defect in kidney during late development

useful technique to study genes with multiple functions

these are conventional techniques

Question 17

What is zinc finger nuclease technology ?

Answer 17

• advance
• method of the year 2011 – nature methods “genome editing with engineered nucleases”
• for its annual choice to highlight an important research method for biological researchers Nature Methods has selected “genome…” see Nat methods. 2011 Dec 28; 9(1):26
• in 2011 there were many high profile publications using zinc finger nucleases wiht a wide range of genomic edits in a variety of cell lines and species

zinc finger binding motif: zinc fingers are a type of protein structural motif that binds to DNA e.g. there are also leucine zippers, helix-loop-helix binding domains etc

• binds to DNA at three points
• important: specific amino acids on the proteins are specific for DNA sequences i.e. proteins do not bind randomly
• create two x two zinc finger proteins each with specificity for 12 nucleotides
• to each of the two finger motifs, attach a DNA nuclease - Fok 1
• when the DNA-binding and DNA-cleaving domains are fused together, a highly-specific pair of ‘genomic scissors’ are created
• a break can be made in a specific location anywhere in the genome
• can target where you want to make break in DNA by changing nucleotides on zinc finger proteins cDNA
• use expression vector to get zinc finger nuclease (ZFN) gene into cell of interest
• ZFN pair recognise and bind to target site
• ZFN makes double strand break
• the break stimulates DNA repair
  a) 1-20% of cells are mis-repaired resulting in gene deletion
  b) when repair template co-transfected with ZFN pair, 2-20% cells contain GOI at target site via homologous recombination

Question 18

What are advantages of ZFN technology?

Answer 18

• For A) deletion mutation at, or B) integration into, any genomic location
• change is permanent and heritable
• works in many mammalian cells
• no selectable markers needed

Question 19

what is ZFN gene-targeting technique?

Answer 19

• fertilised one-cell embryo
• microinjection of ZFN into nucleus
• ZFN causes disruption of targeted gene

1. transfer of modified embryos to foster mothers
   - birth of founder animals

or

2. culture-growth of modified embryo (e.g. zebrafish)
   - birth of founder animals

Question 20

What are knockout models currently availabe?

Answer 20

- neurodegenerative disorders in rats 
parkinson's
autism (Fmr1, Nlgn3) 
schizophrenia 
alzheimer's disease